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* Laboratory of Molecular Genetics, Istituto Giannina Gaslini, Genova, Italy;
Advanced Biotechnology Center, Genova, Italy;
Unita Operativa Medical Genetics, Policlinico Orsola-Malpighi, Bologna, Italy;
Functional Genomics, National Cancer Research Institute, Genova, Italy; and
¶ Laboratorio Centrale di Analisi, Istituto Giannina Gaslini, Genova, Italy
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Abstract |
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionDisclosuresReferences |
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Anion transport is regulated by acute and chronic stimuli. Acute activation of Cl– transport is triggered by signals that elevate intracellular cAMP or Ca2+ (2). The increase of cAMP activates CFTR through phosphorylation of its R domain (8). In contrast, Ca2+ elevation causes the activation of Ca2+-activated Cl– channels, a different class of membrane proteins whose identity is controversial (7). Chronic regulation seems also important as demonstrated by experiments on cultured cells where treatment with IL-4/IL-13 causes an up-regulation of Cl– transport, particularly of the Ca2+-dependent component (9, 10).
It has been recently postulated that SCN– (thiocyanate) transport may also play a very important role in the pathophysiology of the airway epithelium (11). SCN– is a pseudohalide normally present in the blood at 30–100 µM and secreted in saliva and milk at concentrations up to 0.5–1.6 mM (12, 13, 14). In such fluids, SCN– has an antimicrobial role (15, 16, 17). Indeed, it is oxidized by H2O2, a reaction catalyzed by lactoperoxidase, to produce OSCN– (hypothiocyanite), a molecule with bactericidal or bacteriostatic activity (18). Recent studies have shown that: 1) lactoperoxidase is secreted on the airway surface (19, 20); 2) the airway epithelium has the ability to generate H2O2 at the apical membrane through dual oxidases 1 and 2 (DUOX1 and DUOX2) (21, 22, 23); 3) polarized preparations of airway epithelium transport SCN– from the basolateral to the apical side in a way that involves CFTR (11); CF cells show a defective bacterial killing due to lack of SCN– transport (24). Such observations suggest that the lactoperoxidase/H2O2/SCN– antimicrobial system is present also in the airways where it could play an important role in innate defense function. They also imply that CFTR dysfunction in CF could favor bacterial colonization by causing a deficit in SCN– transport and that other anion channels and transporters may compensate this deficit by providing an alternate route for SCN–.
In our study, we investigated the mechanisms responsible for SCN– transport in human bronchial epithelial cells under resting and cytokine-stimulated conditions to assess the involvement of CFTR and that of other transport mechanisms. We have found that bronchial epithelial cells possess at least three mechanisms for SCN– transport. Particularly, interesting is the finding that IL-4 causes a dramatic up-regulation of an electroneutral transport for Cl– and SCN–. Global analysis of gene expression by microarrays and functional studies indicate that this SCN– transport is mediated by pendrin (25), an anion transporter whose expression/function in the respiratory system has not been previously reported. Our findings indicate a novel function for pendrin and anion channels in the innate defense of mucosal surfaces.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionDisclosuresReferences |
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FRT cells were cultured in Coon’s-modified F12 culture medium supplemented with 10% FCS. Human bronchial epithelial cells were collected and cultured as previously described (26). Briefly, dissected bronchi were placed overnight at 4°C in a Hanks’ solution containing protease XIV. The bronchi were then gently removed from the protease solution, and epithelial cells were collected by flushing energically the bronchial lumen with Hanks’ solution. Detached epithelial cell layers were pelleted by centrifugation and resuspended in a small volume of saline solution containing trypsin. After 5–10 min at 37°C, single cells were dissociated by repeated pipetting. After this step, trypsin was neutralized with a culture medium containing serum. Cells were then pelleted by centrifugation and plated in culture flasks in a serum-free culture medium consisting in a 1:1 mixture of LHC9 and RPMI 1640 (26). After two to five passages, cells were frozen in several aliquots. When needed, a single aliquot of bronchial cells was thawed and cultured for 1–2 more passages in the serum-free medium. At the end, the cells were plated at high density (500,000 cells/cm2) on Snapwell (12-mm diameter) or Transwell (24.5-mm diameter) porous supports (code 3801 and 3450, respectively; Corning Costar). After 24 h, the serum-free medium was replaced with an enriched mixture containing DMEM/F12 (1:1) plus 2% FCS and various hormones and supplements (26). Generation of differentiated epithelia was checked by measuring daily transepithelial electrical resistance and potential difference with an epithelial volt ohmmeter (World Precision Instruments). Experiments were usually done at 10–14 days after plating, when epithelial resistance and potential difference were 1.5–2.5 k
· cm2 and –40 to –50 mV, respectively.
Gene expression analysis by microarrays
Total RNA was extracted using TRIzol reagent (Invitrogen Life Technologies) followed by purification with RNeasy Mini kit (Qiagen) according to the manufacturer’s instructions. cDNA synthesis was performed using T7-(dT)24 oligonucleotide primers and the Custom SuperScript Double-Stranded cDNA Synthesis kit (Invitrogen Life Technologies). Double-stranded cDNAs were extracted with phenol-chloroform-isoamyl alcohol (25:24:1), ethanol precipitated, and used to prepare cRNAs using the Bioarray High Yield RNA Transcription kit (Affymetrix) according to the manufacturer’s instructions. cRNAs were purified using the RNeasy Mini kit (Qiagen), controlled by agarose gel electrophoresis and RNA 6000 Pico Assay (Agilent Technologies) and subjected to fragmentation for 35 min at 94°C in fragmentation buffer (40 mM Tris-acetate (pH 8.1), 100 mM CH3COOH, 30 mM Mg(CH3COO)2.
Labeled cRNA was used for hybridization of GeneChip Human Genome U133 plus 2 arrays (Affymetrix). The experiment consisted of four biological replicates (different donors) for control and IL-4-treated cells. Hybridization and scanning was conducted on the Affymetrix platform. Data were normalized following the guanine cytosine robust multiarray average procedure (27) of Bioconductor 1.8 (28) (www.bioconductor.org). Normalization was set to "invariant.set." Statistically significant expression changes were determined using permutation tests, significance analysis of microarrays (29) (www-stat.stanford.edu/
tibs/SAM/) considering treated and untreated cells from the same donor as a pair. Genes regulated at least 2-fold in comparison to untreated controls were considered. The 
value was set to return a median false significant number <1. Annotations were obtained through the DAVID database (http://david.niaid.nih.gov/david/
/index.htm) (30).
Gene expression analysis by RT-PCR
For real-time quantitative PCR, 1 µg of total RNA was retrotranscribed with both random hexamer and oligo(dT) primers using the Advantage RT-for-PCR kit (BD Clontech). Quantitation of transcripts for desired genes was conducted using inventoried Assays-on-Demand provided by Applied Biosystems (specifically: Hs001166504_m1 for pendrin, and Hs00187842_m1 for 2-microglobulin as endogenous control). PCR was performed using the ABI Prism 7700 Sequence Detection System (Applied Biosystems). Reaction conditions were: 2 min at 50°C for UNG activation and 10 min hot start at 95°C, followed by 40 cycles, each consisting in denaturation at 95°C for 15 s, and annealing/extension at 60°C for 1 min. Changes in transcript levels were quantified by using the comparative cycle threshold method (Sequence Detection System Chemistry Guide; Applied Biosystems). Each sample was run in triplicate. Data were analyzed by using the Sequence Detector Systems version 2.0 software (Applied Biosystems).
Cloning of human pendrin
Total RNA extracted from bronchial epithelial cells was retrotranscribed to cDNA as described above. Amplification of full-length pendrin coding sequence was obtained in a reaction containing: 7 µl of bronchial cell cDNA, 2 mM Mg2+, 20 µM dNTPs, 1 µM forward and reverse primers, 0.25 µl of AmpliTaq Gold (PerkinElmer), in a final volume of 25 µl. Forward primer sequence was: 5'-CCACTGCCTTCTGAGAGC-3'. Reverse primer sequence was: 5'-CCTAGAAGCAGTCTTAGTGC-3'. The PCR consisted in a first step at 95°C for 12 min, followed by 35 cycles of 95°C for 30 s, 60°C for 30 s, 72°C for 2 min, and a final step of 72°C for 7 min. The amplification product was cloned in the pcDNA3.1 TOPO vector (Invitrogen Life Technologies) following the manufacturer’s instructions. The resulting plasmids extracted from bacterial colonies were analyzed with SacII digestion to identify the constructs with the correct orientation of the insert. Full sequencing of the insert was then conducted to confirm identity of the cloned product with human pendrin (SLC26A4).
Transfection in FRT cells and analysis of anion transport by fluorescence
The plasmid vector carrying pendrin-coding sequence and neomycin-resistance gene was transfected in FRT cells already expressing the yellow fluorescent protein (YFP)-H148Q/I152L by means of the Lipofectamine 2000 reagent (Invitrogen Life Technologies). Cells were then treated with 0.75 mg/ml G418 and 0.5 mg/ml hygromycin B to select clones with stable coexpression of pendrin and the YFP. Determination of pendrin activity was conducted by plating the cells in 96-well microplates (50,000 cells/well). After 24 h, the cells were washed three times with 200 µl of PBS (in mM: 137 NaCl, 2.7 KCl, 8.1 Na2HPO4, 1.5 KH2PO4, 1 CaCl2, 0.5 MgCl2). After washing, cells were left for 15–30 min at 37°C in 60 µl of PBS per well. The microplate was then transferred to a BMG Fluostar Galaxy plate reader equipped with excitation and emission optical filters for YFP (excitation: HQ500/20X, 500 ± 10 nm; emission: HQ535/30 M, 535 ± 15 nm; Chroma) and injection pumps. The assay consisted, for each well, in the continuous reading of fluorescence for 14 s. At 2 s from start, the plate reader injected 165 µl of a modified PBS in which Cl– was replaced by I–, SCN–, NO3–, or Br–. The decrease of cell fluorescence caused by addition of quenching anions was analyzed to calculate anion transport. Briefly, the fluorescence decay phase was fitted with an exponential function to derive the maximal slope. Maximal slopes were converted to rates of variation of intracellular anion concentration (in millimoles per second) using the equation: d[X–]/dt = KX[d(F/F0)/dt], where [X–] is the anion concentration, KX is the affinity constant of YFP for a given anion, and F/F0 is the ratio of the cell fluorescence at a given time vs initial fluorescence. Activity in cells transfected with pendrin was compared with that in null cells or in cells expressing CFTR. Before the assay, CFTR was preactivated with 20 µM forskolin.
Transepithelial SCN– and Cl– transport
FRT cells (with and without expression of pendrin or CFTR) or bronchial epithelial cells were plated on Transwell permeable supports (500,000 cells/cm2) and cultured with the appropriate medium (1.5 ml on the apical side, 2.5 ml on the basolateral side). After 10–12 days, the Transwell inserts with cells were washed on both sides with PBS and then transferred to a 6-well plate seating on the top of metal block heated at 37°C. Three Transwell inserts were processed at each time. Each well (basolateral side) contained 2 ml of PBS plus 10 mM glucose and 0.4 µCi of 36Cl– or S14CN– (total concentration of SCN–: 95 µM). The top of the Transwell (apical side) instead received 1 ml of PBS. For bronchial cells, the apical solution also contained 10 µM amiloride to block the epithelial sodium channel. After incubation of cells for 15 min, the apical fluid was removed and replaced with 1 ml of the same solution preheated at 37°C. This process was repeated every 2 min. The apical fluid collected in the first three time points was discarded. Subsequently, the fluid collected every 2 min was placed in scintillation vials for radioactivity determination.
Short-circuit current recordings
Bronchial epithelial cells or FRT cells plated on Snapwell supports were mounted in a self-contained Ussing chamber system (vertical diffusion chamber; Corning Costar). FRT cells were studied with a transepithelial Cl– gradient. Accordingly, the basolateral solution contained (in millimoles): 130 NaCl, 2.7 KCl, 1.5 KH2PO4, 1 CaCl2, 0.5 MgCl2, 10 sodium-HEPES (pH 7.3) and 10 glucose. For the apical side, this solution was instead modified by replacing half of NaCl with sodium gluconate and increasing CaCl2 to 2 mM to compensate for calcium buffering caused by gluconate. The basolateral membrane was permeabilized with 250 µg/ml amphotericin B. For, human airway epithelial cells, both apical and basolateral chambers contained (in millimoles): 126 NaCl, 0.38 KH2PO4, 2.1 K2HPO4, 1 MgSO4, 1 CaCl2, 24 NaHCO3, and 10 glucose (basolateral membrane not permeabilized). During experiments, solutions in both chambers were continuously bubbled with air (FRT cells) or with 5% CO2 in air (bronchial cells). The hemichambers were connected to DVC-1000 voltage clamps (World Precision Instruments) via Ag/AgCl electrodes and 1 M KCl agar bridges. Transepithelial currents were digitized using PowerLab 4/25 and 2/25 data acquisition systems and stored on Macintosh computers. All measurements were done at 37°C.
Immunodetection of pendrin protein
Bronchial epithelial cells or FRT cells plated on Snapwell supports were fixed in paraformaldehyde (4%) and permeabilized with Triton X-100 (0.2%). Fixed epithelia were then incubated with anti-pendrin Abs (31) at a concentration of 0.25 µg/ml for 2 h at 37°C. After washings with PBS, cells were incubated with a Cy3-labeled anti-rabbit secondary Ab (Molecular Probes). At the end, the porous membrane with the stained epithelium was detached from the plastic support and mounted on a slide using the VectaShield mounting medium with 4',6'-diamidino-2-phenylindole (Vector Laboratories). Photographs were acquired with a fluorescence microscope equipped with a digital camera and the IPLab software.
Transfection with short-interfering RNA (siRNA)
Previous unpublished studies from our laboratory have shown that best conditions for siRNA transfer to bronchial epithelial cells require transfection at the time of cell plating on permeable supports. Accordingly, bronchial cells were coplated on the apical side of Transwell inserts in 2 ml of LHC9/RPMI 1640 medium without antibiotics together with 500 µl of OPTI-MEM medium (Invitrogen Life Technologies) containing preformed complexes of siRNA (20–100 nM final concentration) and 20 µl of Lipofectamine 2000 (Invitrogen Life Technologies). Stealth RNA interference against human pendrin (code HSS107795) and corresponding negative control (code 12935-200) were from Invitrogen Life Technologies. The anti-pendrin siRNA was chosen as the one inducing the most effective silencing in FRT cells expressing human pendrin. The negative control was a nontargeting siRNA having a guanine cytosine content matching that of the antipendrin duplex. During transfection, the basolateral side contained 2.5 ml of LHC9/RPMI 1640 medium without antibiotics. siRNA complexes were removed after 24 h. Transepithelial SCN– transport was conducted after 10–12 days.
Measurement of H2O2
Generation of H2O2 was measured with the Amplex Red Hydrogen Peroxide/Peroxidase Assay kit (Molecular Probes/Invitrogen Life Technologies). Bronchial epithelial cells were plated and cultured on Snapwell permeable supports as for short-circuit current recordings. After 10–14 days, the Snapwell supports containing the cells were washed with PBS and positioned in a 6-well plate, four supports each time. The well (basolateral side) contained 2 ml of PBS plus 10 mM glucose. The top of the Snapwell (apical side) instead received 250 µl of PBS without glucose. After 20 min of equilibration at 37°C, 200 µl of the apical solution were replaced with an equal volume of PBS containing Amplex Red reagent and HRP, to a final concentration of 25 µM and 0.1 U/ml. After addition of reagents, measurement of H2O2 started immediately by reading fluorescence with a BMG Fluostar Galaxy plate reader. The reader was programmed to read fluorescence from each one of the four Snapwell supports every 6 s for 10 min. Excitation and emission wavelengths were 544 and 590 nm, respectively. After this step, UTP (final concentration 100 µM) was added to the apical solution and fluorescence reading was continued for further 10 min.
Intracellular-free Ca2+ detection
Bronchial epithelial cells, cultured on Snapwell supports, were placed in a six-well plate after washing apical and basolateral sides with PBS. The well (basolateral side) contained 2 ml of PBS plus glucose 10 mM. The apical side of the Snapwell support contained instead 200 µl of PBS. Cells were incubated for 1 h at 37°C with 4 µM Fluo-4/AM (Molecular Probes) and 2 mM probenecid. After Fluo-4 loading, cells were washed leaving 200 µl and 2 ml of PBS plus probenecid in the apical and basolateral sides, respectively. The multiwell plate containing the Snapwell supports was then transferred to the microplate reader for fluorescence measurement. The assay was run for one Snapwell at a time and consisted in continuous fluorescence reading for 35 s. Excitation and emission wavelengths were 485 and 520 nm, respectively. At 8 s, the plate reader was programmed to inject 330 µl of PBS plus UTP (100 µM final concentration) on the apical side.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionDisclosuresReferences |
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We determined transepithelial Cl– transport in the same conditions and with the same procedure used for SCN–. We found a behavior qualitatively similar to that of SCN– (Fig. 1, C and D). A CFTR-dependent component of Cl– transport (i.e., activation by forskolin and block by CFTRinh-172) was detected in normal but not in CF cells. Furthermore, a significant increase in basal Cl– flux was obtained by incubating the cells with IL-4. Although similar, the data obtained with the two anions differed from the quantitative point of view. The stimulation with IL-4 elicited a larger effect on SCN– compared with Cl– (10- vs 4-fold, respectively). It is also interesting to note that if we normalize for the different concentrations used in the experiments to mimic physiological conditions (95 µM SCN– vs 137 mM Cl– in the basolateral solution), it appears that permeability for SCN–, in particular after IL-4 treatment, is even higher than that for Cl–.
The strong up-regulation of basal anion transport by IL-4, detected by radioisotopic technique, was particularly interesting because we had not detected in previous studies a similar effect during short-circuit current recordings (9), as also shown in Fig. 1G. In agreement with our previous findings, we found that treatment with IL-4 caused a dramatic reduction of Na+ absorption as evident from the reduced effect of the ENaC blocker, amiloride. Upon block of ENaC with amiloride, and of CFTR with CFTRinh-172, we observed that the residual current was very small and not different between control and IL-4-treated cells (Fig. 1G). This contrasts with the high Cl– and SCN– fluxes evoked by IL-4 treatment. Therefore, we hypothesized that the strong effect elicited by IL-4 on basal SCN– and Cl– transport is mediated by stimulation of a nonelectrogenic anion transporter, possibly by up-regulation of transcription of the corresponding gene.
To possibly identify this transporter, we performed a global analysis of gene expression by means of Affymetrix U133 Plus 2.0 microarrays. This last version of microarrays contains 54,000 probe sets corresponding to >47,000 human transcripts. The mRNA was extracted from untreated and IL-4-treated cells of cultured polarized epithelia of four different individuals. We looked for possible up-regulation of anion channels and transporters. We found no up-regulation of Cl– channels of the ClC family like CLC2 (data not shown). The same negative findings were found for putative Ca2+-dependent Cl– channels of the ClCA or bestrophin family. Actually, the genes belonging to the latter family appeared as poorly expressed in our cells (data not shown). Interestingly, the only anion transporter that appeared strongly up-regulated by IL-4 was SLC26A4, also known as pendrin (25) (see Table I). The up-regulation consisted in a 23-fold increase of SLC26A4 signal in cytokine-treated cells. This finding was surprising because pendrin expression in airway epithelial cells has not been previously reported. Pendrin is a transporter which mediates anion exchange at the apical membrane of cells in the kidney, inner ear, and thyroid follicle (34, 35, 36, 37). When mutated, the pendrin gene is responsible for Pendred syndrome or nonsyndromic deafness (38, 39). Besides pendrin, no other known anion transporter was strongly up-regulated by IL-4. Many other members of the SLC26 family, like SLC26A1, SLC26A6, SLC26A7, SLC26A8, SLC26A9, and SLC26A10, were expressed at a low level in our cells and not affected by IL-4. SLC26A2, a sulfate transporter, was the only member of the same family with a significant expression under basal levels and a modest induction by IL-4. Similarly, other anion transporters belonging to the SLC4 family (anion exchangers) were poorly expressed and/or not affected by cytokine treatment. To confirm the findings obtained with the microarrays, we determined the changes in expression of specific genes by real-time RT-PCR. We found that pendrin mRNA was indeed strongly up-regulated after treatment with IL-4 (24.7- ± 4.9-fold, n = 6). Other transporters of the same family, including SLC26A3, A6, A7, A8, and A9 were instead poorly expressed and not up-regulated. We also measured by the same method the expression of SLC5A5, the Na+/I– symporter, which is also able to mediate SCN– uptake (11). This gene was not up-regulated by IL-4 in agreement with data deriving from microarrays (Table I).
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2500 bp, compatible with the expected pendrin amplification product (data not shown). This fragment was cloned in a plasmid and the resulting clones were fully sequenced. Determination of nucleotide sequence revealed that the clones contained the full coding sequence of pendrin, as reported in GenBank (accession number: NM_000441). To determine the functional properties of cloned pendrin we decided to take advantage of the halide and pseudohalide sensitivity of YFPs (40, 41). Accordingly, we transfected the expression vector carrying the pendrin-coding sequence in FRT cells that already expressed the YFP-H148Q/I152L (41). FRT cells are thyroid cells that have lost expression of endogenous pendrin. The resulting cell line coexpressing the two desired proteins was used in fluorescence assays using a microplate reader equipped with syringe pumps for liquid injection during the assay. Injection in the well of a modified PBS containing I– instead of Cl– generated a decrease in fluorescence that was much faster in pendrin-expressing cells compared with null cells (Fig. 2A). This is in agreement with the reported ability of pendrin to mediate exchange of Cl– with I– (25, 39). In the same way, a fast fluorescence quenching was also obtained by injecting a SCN–- or a NO3–-rich solution thus indicating that pendrin is also able to transport these anions (Fig. 2A). We calculated anion fluxes by taking into account the different affinity of the YFP for the different anions (41). After this correction, the rates of transport for the different anions in pendrin-expressing cells were comparable (Fig. 2B). We performed the same type of experiments also in FRT cells expressing CFTR. In this case, we found that I– and SCN– transport rates were lower than those measured for NO3– and Br– (Fig. 2C). This is in agreement with the CFTR pore being partially blocked by such anions (42).
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We looked for pendrin protein expression by immunofluorescence using Abs directed against pendrin N and C termini (Fig. 5). Immunostaining was clearly detected with either Ab when bronchial epithelial cells were treated with IL-4. A similar signal was also detected in FRT cells expressing human pendrin whereas untransfected cells were indistinguishable from cells incubated with second Ab alone (data not shown).
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. In fact, preliminary results with microarrays have shown that this cytokine also induces pendrin expression (our unpublished results). We have conducted real-time PCR to evaluate relative pendrin expression in treated and untreated cells. We found that treatment for 24 h with 2.5 ng/ml IL-1 caused a 23-fold induction of pendrin mRNA. Consequently, we performed transport studies in IL-1-treated cells. We found that treatment with this cytokine caused a significant increase in SCN– transport which was affected by removal of apical Cl– as in IL-4-treated cells (Fig. 6A). In the second approach, we directly attempted to silence pendrin mRNA with siRNA. siRNA against pendrin (20–100 nM) was transfected in bronchial epithelial cells at the time of cell plating. After generation of the polarized epithelium, cells were treated with IL-4. We found that treatment with anti-pendrin siRNA generated a dose-dependent decrease of SCN– transport with respect to cells transfected with a nontargeting siRNA (Fig. 6B). Maximum inhibition of SCN– transport (50–60%) was obtained with 100 nM siRNA. By real-time RT-PCR, we found that pendrin mRNA was 80% decreased in cells treated with specific pendrin siRNA (100 nM) compared with negative control at the same concentration (data not shown). We found no effect of transfection reagent alone on either anion transport or pendrin mRNA.
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The physiological meaning of SCN– transport across airway epithelium is probably to provide one of the substrates for lactoperoxidase, the other substrate being H2O2. We asked whether up-regulation of SCN– transport in IL-4-treated cells is paralleled by a change in the ability to generate H2O2. We measured the production of H2O2 on the apical membrane of bronchial epithelial cells using the Amplex Red fluorescent reagent. Under resting conditions, we detected a significant generation of H2O2, whose rate was markedly accelerated by addition of UTP (Fig. 8A). This stimulation is considered to be the consequence of intracellular Ca2+ increase and activation of dual oxidases (DUOX1 and DUOX2), the membrane enzymes responsible for extracellular H2O2 production (21, 47). Interestingly, cells pretreated with IL-4 showed an up-regulation of H2O2 release, particularly of the UTP-stimulated component. H2O2 production was strongly inhibited in the presence of 20 µM diphenyleneiodium, an inhibitor of dual oxidases (data not shown). Examination of microarray data revealed that both DUOX1 and DUOX2 transcripts are increased (2- to 3-fold) by IL-4 treatment (Table I). These data were confirmed by real-time RT-PCR.
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We asked whether the rate of SCN– transepithelial transport was adequate to sustain H2O2 conversion by lactoperoxidase. Accordingly, we mimicked the physiological situation by measuring H2O2 in the presence of apical lactoperoxidase and basolateral SCN–. We found that the amount of detectable H2O2 on the apical side was strongly decreased when both SCN– and lactoperoxidase were present during the experiment, but unchanged with lactoperoxidase alone (Fig. 8, C and D).
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Discussion |
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Interestingly, our study demonstrates for the first time that the bronchial epithelium expresses also other pathways for SCN– transport in addition to CFTR. These alternative pathways are particularly sensitive to regulation by IL-4. In a previous study, we reported that IL-4 has a pleiotropic effect on transepithelial ion transport in bronchial epithelial cells (9). In particular, we found that anion transport is in general stimulated by the cytokine. We interpreted this effect by postulating that IL-4 stimulates Cl– secretion as a way to improve airway surface hydration and mucociliary clearance. Given the importance of SCN– in host defense, we have now tested the possibility that SCN– transport is also stimulated by IL-4. We have found that cells treated with IL-4 show a marked increase in transepithelial SCN– flux. This type of response is independent of CFTR because it is preserved in CF cells and is not blocked by CFTRinh-172. Actually, our data indicate that the SCN– transport induced by IL-4 is mediated by an electroneutral transporter which exchanges SCN– with Cl–. We hypothesized that the mechanism of IL-4 activity is based on the increased expression of a membrane SCN–/Cl– transporter. Using microarray analysis of gene expression, we have found that the only known transporter that appears strongly up-regulated in IL-4-stimulated cells is pendrin (SLC26A4). The cytokine effect consists in a >20-fold elevation of pendrin signal. Expression of cloned pendrin in FRT cells induced the appearance of a SCN– transport whose properties (e.g., affinity for Cl–) were similar to those observed in bronchial epithelial cells treated with IL-4. Besides these observations, other three lines of evidences are in support of pendrin involvement in cytokine-stimulated SCN– transport. First, we treated bronchial cells with IL-1. This treatment also resulted in up-regulation of either SCN– transport and pendrin mRNA. Second, treatment with IL-4 caused the appearance of immunostaining of bronchial cells with a pendrin Ab. Third, treatment with an anti-pendrin siRNA caused a significant reduction in SCN– transport.
We investigated whether SCN– transport is also mediated by Ca2+-dependent Cl– channels. To this aim, we measured the response of bronchial cells to UTP. This nucleotide induces transient activation of Ca2+-dependent Cl– channels through P2Y2 purinergic receptors (46). We hypothesized that this activation could result in increased SCN– transport, because Cl– channels generally allow transport of various anions besides Cl– (7). We found that UTP really stimulated transepithelial SCN– transport. The response was transient, with a fast peak followed by a progressive decay that ended in a few minutes, a behavior that reproduced the time course of the Cl– secretion activated by UTP in short-circuit current conditions. Furthermore, the UTP-dependent SCN– transport was up-regulated in IL-4-treated cells, by an extent similar to that of UTP-dependent Cl– secretion, as expected if both processes occur through the same channels. The purinergic-activated transport was not affected by removal of apical Cl–, thus showing that it is different from the electroneutral SCN– transport induced by IL-4. In addition, the up-regulation of Ca2+-dependent anion channels by IL-4 does not depend on a modification of the Ca2+ signal because in control and cytokine-treated cells UTP triggered a similar Ca2+ increase. Therefore, the mechanism of IL-4 lies in a step downstream of the Ca2+ increase, possibly involving the channels themselves or channel regulators.
It is interesting to point out that we found also an increase in the ability to produce H2O2 after treatment with IL-4. This may reflect the up-regulation of DUOX1 and DUOX2 transcripts as evidenced by microarray data. A recent report also indicates that DUOX genes transcription is affected by cytokines (48).
In conclusion, we have identified three mechanisms for SCN– transport in bronchial epithelial cells (Fig. 9). One mechanism involves CFTR as indicated by previous findings (11, 24). Lack of this transport could be important in the pathogenesis of CF lung disease. In addition, we have found that an alternative pathway for SCN– transport involves Ca2+-dependent anion channels. Such channels are considered as an important pharmacological target to overcome the CFTR defect in CF patients (49). Indeed, a purinergic agonist, deflunosol, is in clinical trials to demonstrate the efficacy in the improvement of lung function in CF patients (50). The hypothesis is that stimulation of Cl– secretion through Ca2+-dependent channels would improve mucociliary clearance. Our findings, by showing that UTP also stimulates SCN– transport, could further enhance the relevance of purinergic stimulation as a therapeutic strategy in CF. Indeed, this approach would increase the availability of apical SCN–, which seems to be defective in CF patients due to CFTR loss of function. The finding that the purinergic signal regulates SCN– transport is particularly interesting. Because H2O2 production is also dependent on purinergic regulation, it is possible to envision a coordinated response where local activation of receptors may enhance antimicrobial activity by providing the two substrates needed by lactoperoxidase.
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Various intriguing questions arise about the physiological meaning of SCN– transport and the up-regulation by IL-4. SCN– reacts with H2O2, a reaction catalyzed by lactoperoxidase, to generate OSCN–, a bacteriostatic/bactericidal molecule (18). Hence, up-regulation of SCN– transport by IL-4, and possibly by other cytokines like IL-1, may be considered as a mechanism to potentiate antimicrobial activity. However, it has to be noted that H2O2 has a bactericidal activity by itself, but also a toxicity to epithelial cells, stronger than OSCN–. Therefore, SCN– transport may also be important to scavenge excessive H2O2 generation under particular inflamed conditions. In addition, it has been reported that SCN– is also a substrate for neutrophil and eosinophil peroxidases (myeloperoxidase and eosinophil peroxidase, respectively) (51, 52, 53). Actually, availability of SCN– in the airway periciliary fluid may switch the activity of cellular peroxidases toward the production of OSCN– instead of OCl– and OBr–, which are highly cytotoxic oxidants. Accordingly, up-regulation of transepithelial SCN– transport by IL-4 could be a way to scavenge oxidants and to dampen the toxicity arisen from cells like eosinophils and neutrophils that are recruited in the airway lumen during inflammatory conditions.
Future studies will have to determine which of the possible effects of IL-4-stimulated SCN– transport (i.e., antimicrobial vs antioxidant) is most important. In addition, it will be interesting to assess whether and how SCN– transport through non-CFTR channels and transporters affects the lung disease in CF patients. There is a large variability in the severity of lung pathology in CF which is attributed to the role of other proteins in addition to CFTR. Genes involved in the function of the lactoperoxidase/SCN–/H2O2 system like SCN– transporters may be important as modifiers of the CF phenotype in the lung. Regarding pendrin, the presence of cytokines in inflamed airways may lead to expression of this transporter with a possible compensation of the defective SCN– transport in CF airways and partial recovery of antimicrobial activity. However, this hypothesis needs to be verified in vivo because inflamed airways contain a complex mixture of soluble mediators whose net on pendrin expression cannot be anticipated. Another intriguing question regards the absence of a clear respiratory disease in individuals affected by mutations in the pendrin gene (which cause Pendred syndrome or nonsyndromic deafness). It is possible that absence of pendrin function in the lung of such patients is compensated by the expression of other transporters. In addition, it is possible that deficit of pendrin function modulates the severity of acute/chronic pathologies of the lung in a way that has not been detected so far.
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1 This work was supported by grants from Telethon-Italy (GGP05103), the Cystic Fibrosis Foundation Therapeutics, Comitato Interministeriale Programmazione Economica Regione Liguria (Biofarma 2), and the National Institutes of Health (P30 DK072517). 
2 Address correspondence and reprint requests to Dr. Luis J. V. Galietta, Laboratorio di Genetica Molecolare, L.go Gerolamo Gaslini 5, 16147 Genova, Italy. E-mail address: galietta{at}unige.it
3 Abbreviations used in this paper: CF, cystic fibrosis; FRT, Fischer rat thyroid; siRNA, short-interfering RNA; YFP, yellow fluorescent protein; CFTR, cystic fibrosis transmembrane conductance regulator; PDS, pendrin.
Received for publication September 12, 2006. Accepted for publication February 1, 2007.
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) or treated with IL-4 (
). G, representative traces from Ussing chamber experiments on resting (top) and IL-4 treated (bottom) bronchial epithelial cells. Except for Ussing chamber experiments, all data are presented as mean ± SEM of four to six experiments. ***, A statistically significant difference (p < 0.001).
). Each point is the mean ± SEM of four experiments. A significant reduction of SCN– transport (<0.01) was obtained at all time points using 100 nM siRNA, and for the last two time points for 50 nM siRNA.
